N. Ronald Morris

A simplified ultrasensitive silver stain for detecting proteins in polyacrylamide gels

Abstract We have simplified the highly sensitive silver stain of R. C. Switzer III, C. R. Merril, and S. Shifrin (1979, Anal. Biochem.98, 231–237) for visualizing proteins in polyacrylamide gels. We have reduced the number of steps in the procedure from 10 to 6, simplified the reagents in each step, and reduced the amount of silver required by a factor of 10, thus greatly reducing the expense of the procedure. In common with the original silver stain, our procedure is 100 times more sensitive than Coomassie brilliant blue and is comparable in sensitivity to radioautography of radioactively labeled proteins.

Mutation of a gene that encodes a kinesin-like protein blocks nuclear division in A. nidulans

In A. nidulans, the temperature-sensitive cell cycle mutation bimC4 causes an elevated mitotic index at restrictive temperature. Under restrictive conditions the mutation interferes with separation of the spindle pole bodies, causes abnormal spindle morphology, and prevents nuclear division. We have cloned and sequenced the wild-type bimC gene. The predicted protein product has homology to Drosophila kinesin heavy chain. We conclude that this kinesin-like protein has an important role in nuclear division in Aspergillus.

Characterization of an inducible expression system in Aspergillus nidulans using alcA and tubulincoding genes

Plasmids have been constructed in which expression of a gene can be placed under the control of the inducible promoter of the alcA gene encoding alcohol dehydrogenase I in Aspergillus nidulans. Simplified shuttle vectors carrying pyr4 which complements pyrG89 mutations have also been constructed. These are based on pUC19 and retain alpha-peptide expression. The beta-tubulin genes, tubC and benA, have been placed under the control of alcA and their expression studied. Levels of expression can be assayed phenotypically because increased synthesis of beta-tubulin inhibits vegetative growth. Sensitivity of asexual spore formation to the anti-microtubule drug benomyl provides a means of detecting very low levels of expression of the chimeric genes. Glucose almost completely represses the chimeric genes. Induction is rapid and is maximal within an hour. When a strain carrying seven copies of an alcA::tubC gene fusion was grown under inducing conditions, 6.5% of total sulfate labelled protein consisted of tubC product. Cyclopentanone was the most potent inducer of the chimeric genes on solid media but it also partially inhibited growth. Chimeric alcA::tubC and alcA::benA genes were expressed to very similar levels despite the fact that tubC utilizes many rare codons.

Identification of a gene for β-tubulin in aspergillus nidulans

N. Ronald Morris, Margaret H. Lai and C. Elizabeth Oakley Department of Pharmacology CMDNJ-Rutgers Medical School Piscataway, New Jersey 08854 Summary This paper demonstrates that revertants of tem- perature-sensitive benA (p-tubulin) mutations in Aspergillus nidulans can be used to identify pro- teins which interact with /3-tubulin. Three beno- myl-resistant benA (P-tubulin) mutants of Asper- gillus nidulans, BEN 9, BEN 15 and BEN 19, were found to be temperature-sensitive (ts-) for growth. Temperature sensitivity co-segregated with benomyl resistance among the progeny of outcrosses of BEN 9, 15 and 19 to a wild-type strain, FGSC#99, indicating that temperature sensitivity was caused by mutations in the benA gene in these strains. Eighteen revertants to ts+ were isolated by selection at the restrictive tem- perature. Four had back-mutations in the benA gene and fourteen carried extragenic suppressor mutations. Two of the back-mutated strains had p-tubulins which differed from the p-tubulins of their parental strains by one (l-) or two (2-) negative charges on two-dimensional gel electro- phoresis. Although the P-tubulins of the extra- genie suppressor strains were all electrophoreti- tally identical to those of the parental strains, one of the suppressor strains, BEN 9R7, had an electrophoretic abnormality in al-tubulin (1). A heterozygous diploid between this strain and a strain with wild-type al-tubulin was found to have both wild-type and mutant (l+) rul-tubulins. This experiment rules out post-translational modifica- tion as a possible cause of the al-tubulin abnor- mality. Thus the suppressor mutation in BEN 9R7 must be in a structural gene for al-tubulin. We propose that this gene be designated tubA to denote that it is a gene for cul-tubulin in A. nidulans. Introduction The assembly of tubulin into microtubules involves it in a large number of a cellular processes. Al- though a great deal is known about microtubules both morphologically and biochemically, there are large gaps in our knowledge. For example, micro- tubule assembly and dynein-dependent microtu- bule-associated movement have been extensively studied in vitro, but almost nothing is known about the regulation of microtubule assembly, disassem- bly or microtubule-dependent transport in the liv- ing cell (Snyder and McIntosh, 1976); and although the molecular architecture of microtubules has been the subject of elegant ultrastructural investi- gation (Snyder and McIntosh, 1976), the molecular details of the interactions between LY- and P-tubu- lins and microtubule-associated proteins are un- known. The genetics of tubulin and microtubules have also not been explored. This paper demon- strates that genetic studies can be used to provide information about the interactions between micro- tubule proteins. Mutants of the fungus Aspergillus nidulans se- lected for resistance to the antimitotic benzimida- zole compounds benomyl or thiabendazole may carry a mutation at any one of three loci, benA, B or C (Van Tuyl, 1977). In a previous paper, we showed that most benA mutants have electropho- retically abnormal p-tubulins and that benA is a structural gene for P-tubulin in A. nidulans (Sheir- Neiss, Lai and Morris, 1978). The present paper extends the potential usefulness of the benA sys- tem for analyzing tubulin structure and function by isolating and characterizing a number of revertants and suppressors of benA mutations. Since the original benA mutants were drug-resistant, it was not possible to isolate revertants directly, but be- cause most of the mutants studied electrophoreti- tally appeared to have missense mutations in the benA gene and because missense mutations may result in destabilization of protein structure, we tested a series of benA mutants for their ability to grow at elevated temperatures with the expectation of finding one or more temperature-sensitive (ts-) strains. Three such ts- mutants were found and characterized, from these a series of revertants to ts+ have been isolated. Among 18 revertants from ts- to ts+, four have intragenic back-muta- tions in p-tubulin and 14 have extragenic suppres- sor mutations. One of the extragenic suppressor strains has electrophoretically abnormal al-tubu- lit-r. The suppressor mutation this strain has been identified as a structural gene for cul-tubulin in A. nidulans. Results BEN 9,155 and 19 Are Temperature-Sensitive Twenty six benA mutants of A. nidulans originally isolated by Van Tuyl (1977) were tested for inability to grow at high temperature. The three strains BEN 9, 15 and 19, carrying the mutations benAll, benA and benA21, were found to be tempera- ture-sensitive (ts-) for growth at 44°C. At this tem- perature, the wild-type parental strain B3 and other benA mutants grew about half as rapidly at 32”C, whereas the three ts- strains were inhibited by -90% (Figure 1). To determine whether the tem-

Spindle formation and chromatin condensation in cells blocked at interphase by mutation of a negative cell cycle control gene

In Aspergillus nidulans the temperature-sensitive cell cycle mutation bimE7 causes chromosome condensation and pre-anaphase spindle formation to occur at restrictive temperature. By constructing double mutants between bimE7 and S phase or G2 phase mutants and blocking DNA replication with hydroxyurea, we demonstrate that bimE7 can cause chromatin condensation and spindle formation in cells held in S or G2. Thus bimE7 overrides normal control systems that prevent mitosis from prematurely occurring during S or G2. We show that bimE7 is a loss of function mutation and propose that bimE normally functions to negatively control a positive mitotic inducing factor, possibly the cell cycle gene nimA.

Nuclear movement is β-tubulin-dependent in Aspergillus nidulans

Abstract We have examined nuclear transport in Aspergillus nidulans to determine whether microtubles function in the movement of this organelle. Nuclear movement was found to be inhibited in germinating conidia (uninucleate asexual spores) by the microtubule inhibitor benomyl. To show that the benomyl inhibition was due to its effect on microtubules, the test was repeated with mutants which have genetic lesions in β-tubulin which produce resistance to benomyl in one case ( ben A15) and super-sensitivity in another ( ben A16). Nuclear movement was resistant to benomyl in the strain carrying ben A15 and super-sensitive in the strain carrying ben Al6. Since altered sensitivity to benomyl in these strains is specifically due to alterations in β-tubulin, these results show that β-tubulin is involved in nuclear movement. To eliminate the possibility that nuclear movement blockage was a secondary consequence of nuclear division blockage, this experiment was repeated with temperature-sensitive nuclear division mutants. At restrictive temperature, nuclear division was blocked in these mutants but nuclear movement was not. In the presence of benomyl, nuclear division and migration were blocked at permissive and restrictive temperatures. Thus nuclear division blockage alone is not sufficient to block nuclear movement. These experiments were corroborated by similar experiments on a temperature-sensitive nuclear movement mutant. Five previously isolated nonallelic temperature-sensitive nuclear movement mutants, nud A-E, were analyzed genetically and found not to be allelic to the ben A (β-tubulin) tubA α-tubulin genes.

Biological function for 6-methyladenine residues in the DNA of Escherichia coli K12☆

Abstract A strain of Escherichia coli K12 mutant at the dam † site contains 0.8 mole % 6-methyl adenine as compared to 0.50 mole % in the wild type, and the residual DNA methylation is not due to the K12 modification methylase specified by the hsp genes. The dam-3 mutant is more sensitive to ultraviolet irradiation and to mitomycin C than the wild type and also shows a higher mutability. DNA isolated from the dam-3 mutant contains single-stranded breaks that are amplified in dam-3 polA12 and dam-3 lig-7 double mutants. A function of dam -specified 6-methyl adenine residues in DNA would, therefore, appear to be the protection of DNA from a nuclease(s) that causes the development of breaks. Combination of dam-3 with polA, recA, recB and recC is lethal.

A β-tubulin mutation in Aspergillus nidulans that blocks microtubule function without blocking assembly

We have isolated a heat-sensitive beta-tubulin mutation, benA33, that blocks nuclear division and nuclear movement at restrictive temperature. This blockage demonstrates that the beta tubulin encoded by the benA gene is essential to both processes. The blockage of both processes is suppressed by the alpha-tubulin mutation, tubA1; thus the alpha tubulin encoded by the tubA gene must also be involved in both processes. When benA33 is shifted from a permissive to restrictive temperature, nuclei are blocked in mitosis. Light microscopy of blocked nuclei reveals that benA33 inhibits movement of chromosomes to the poles, and electron microscopy of blocked nuclei shows that they contain apparently normal spindles. Thus benA33 does not block microtubule assembly but, directly or indirectly, blocks microtubule disassembly. BenA33 also confers resistance to several antimicrotubule agents and the heat sensitivity conferred by benA33 is suppressed by each of these agents. We suggest that the simplest explanation for these effects is that benA33 causes a temperature-dependent hyperstabilization of microtubules that blocks chromosomal movement by blocking microtubule disassembly.

Pleiotropic effects of a DNA adenine methylation mutation (dam-3) in Escherichia coli K12

The dam-3 mutation results in a five-fold reduction in the number of 6-methyl-adenine (6-meA) residues in the DNA of E. coli K12 or phage lambda. The DNA of phage fd appears to be devoid of 6-meA when propagated on dam-3 bacteria. The phenotypic differences between dam-3 and dam+ bacteria include: (i) increased free phage in lysogenic dam-3 cultures, (2) increased sensitivity to methyl methanesulfonate (MMS), (3) inviability of dam-3 lex-I strains, (4) lower molecular weight of DNA in dam-3 bacteria in the absence of DNA ligase and (5) increased rate of DNA degradation in dam-3 recA strains.

Dynamics of cytoplasmic dynein in living cells and the effect of a mutation in the dynactin complex actin-related protein Arp1

Cytoplasmic dynein is a minus-end-directed microtubule motor that participates in multiple cellular activities such as organelle transport and mitotic spindle assembly [1]. To study the dynamic behavior of cytoplasmic dynein in the filamentous fungus Aspergillus nidulans, we replaced the gene for the cytoplasmic dynein heavy chain, nudA, with a gene encoding a green fluorescent protein (GFP)-tagged chimera, GFP-nudA. The GFP-NUDA fusion protein is fully functional in vivo: strains expressing only the GFP-tagged nudA grow as well as wild-type strains. Fluorescence microscopy showed GFP-NUDA to be in comet-like structures that moved in the hyphae toward the growing tip. Retrograde movement of some GFP-NUDA comets after they arrived at the tip was also observed. These dynamics of GFP-NUDA were not observed in cells treated with a microtubule-destabilizing drug, benomyl, suggesting they are microtubule-dependent. The rate of GFP-NUDA tip-ward movement is similar to the rate of cytoplasmic microtubule polymerization toward the hyphal tip, suggesting that GFP-NUDA is associated and moving with the polymerizing ends of microtubules. A mutation in actin-related protein Arp1 of the dynactin complex abolishes the presence of these dynamic GFP-NUDA structures near the hyphal tip, suggesting a targeting role of the dynactin complex.